The present invention relates to a serum-free medium for culturing animal cells, a method for culturing animal cells using the medium, and a method for producing a substance by culturing animal cells in the medium.
Methods for artificial production of various proteins and peptides using recombinant DNA techniques have been developed. Also, methods for creating and culturing transformed cells for use in such methods have been studied. Among these transformed cells, mammal-derived cells are used as host cells for recombinant production of human-derived proteins, etc. to be used in pharmaceuticals. Some of mammal-derived cells are used as host cells. One of the most commonly used mammal-derived cells is Chinese hamster ovary (CHO) cells. As to various selection markers, methods for selecting recombinant cell clones using the markers and host cells which are necessary for application of recombinant DNA techniques, genetic deficiencies against which various selection markers function effectively and a large number of sub-cell lines exhibiting auxotrophy associated with such genetic deficiencies have been established. Conventionally, a medium containing serum or protein components separated from serum has been commonly used for culturing mammal-derived cells. Recently, however, a serum-free medium has been positively developed in order to eliminate contaminants in serum such as viruses or pathogenic prions which must not remain in final products, e.g. recombinant proteins.
A possibility of causing similar contamination has been suggested in the use of proteins separated from serum, such as serum albumin, transferrin, fetuin, various peptide hormones and growth factor proteins, as well as proteins or peptides separated from animals such as blood-containing tissues like beef hydrolysate. Thus, development of a serum-free medium which does not contain even such proteins or peptides has been pursued. Specifically, various peptide hormones and growth factor proteins added to a medium have been replaced with highly purified, corresponding recombinant products as much as possible. In addition, various attempts have been made to replace those proteins or peptides separated from animals with those proteins, peptides or lipids separated from non-animal cells.
A serum-free medium now under development is a basal medium supplemented with various peptide hormones and growth factor proteins for the induction or promotion of cell growth. The basal medium contains appropriate quantities of the substances composing the transformed cells to be cultured (specifically, raw materials such as amino acids, precursors of nucleic acids or nucleosides, aliphatic acids, etc. to be used in the biosynthesis of various proteins, peptides, lipids, nucleic acids, etc. in cultured cells), as well as those constituents of cytoplasm and the like which are taken up from the outside of the cells and used as cell constituents/components per se at the time of cell growth and division (e.g., metal elements, phosphates, chloride ions, and vitamins to be used in coenzymes for enzymes).
In addition to the above-mentioned components essential for the induction or promotion of cell growth per se, addition of supplementary components to maintain the cultivation rate at a high level is under review. Further, addition of supplementary components to promote the production of a gene product of interest, notably recombinant protein or the like, in the transformed cells in culture or to maintain such production at a high level is under review. It is expected to propose a serum-free medium having a cultivation ability comparable to that of the conventional serum-containing medium through selection of the above-mentioned supplementary components to be added to the medium and selection of the optimum amounts of addition of such components. In particular, a new proposal of a serum-free medium is awaited which can achieve an ability comparable to that of the conventional serum-containing medium in culturing animal-derived cells, especially CHO cells, commonly used in the recombinant production of human-derived proteins for use in pharmaceuticals. Furthermore, proposal of a method for culturing animal cells, especially CHO cells, using such a new serum-free medium, as well as a method for performing recombinant production of a human-derived protein of interest at a high efficient by such culturing is also desired.
The present invention has been made to solve the above problems. It is an object of the invention to provide a serum-free medium for culturing animal cells.
It is another object of the invention to provide a method for culturing animal cells in the serum-free medium.
It is still another object of the invention to provide a method which, through the cultivation of animal cells in the serum-free medium, yields a substance that is produced by and secreted out of the animal cells.
Toward the solution of the above-mentioned problems, the present inventors have made extensive and intensive researches. As a result, it was found that growth of animal cells, in particular CHO cells, can be achieved in a serum-free medium which is obtained by adding various peptide hormones and growth factor proteins for induction or promotion of cell growth to a basal medium containing appropriate quantities of various components taken up by the cells from the outside and used as cell constituents/components per se. Subsequently, the inventors have optimized the composition of the basal medium in order to maintain the growth rate at a high level. However, the growth rate of animal cells, in particular CHO cells, was still significantly inferior to the growth rate achieved in the serum-free medium described above supplemented with serum or serum-derived proteins, proteolysates, peptides, etc. The present inventors have further found out that a cultivation ability comparable to the growth rate achieved in serum-containing media can be achieved in a serum-free medium by replacing the components of serum-containing media separated from animals (such as serum or serum-derived proteins, proteolysates, peptides) with a specific combination of components derived from plants and components derived from microorganisms which do not exhibit pathogenicity against human or mammals and by selecting optimum amounts of addition of such components. Based on these findings, the present invention has been achieved.
The present invention provides a serum-free medium for culturing animal cells, containing soybean protein hydrolysate and yeast extract. The serum-free medium of the invention may further comprise wheat protein hydrolysate. By using the serum-free medium of the invention, animal cells can be cultured without addition of components separated from animals, such as serum or serum-derived proteins, proteolysates, peptides. The soybean protein hydrolysate may be added at 1-5 g per liter of the medium. The yeast extract may be added at 1-5 g per liter of the medium. In such a case, wheat protein hydrolysate may be added at a rate of 0.5-3 g per liter of the medium. The ratio by weight of the amount of addition of soybean protein hydrolysate to the amount of addition of yeast extract may be in the range from 80:20 to 60:40. In such a case, the amount of addition of wheat protein hydrolysate may come within the range from 5 to 60% of the total weight of the soybean protein hydrolysate and the yeast extract added. By using the serum-free medium of the invention, it is possible to culture animal cells, preferably mammalian cells, more preferably Chinese hamster ovary (CHO) cells. The animal cells may be transformed cells into which a foreign gene has been transferred.
The present invention also provide a method for culturing animal cells, comprising a step of culturing animal cells in a serum-free medium containing soybean protein hydrolysate and yeast extract. The animal cells are preferably mammalian cells, more preferably CHO cells. The animal cells may be transformed cells into which a foreign gene has been transferred.
Further, the present invention provides a method for producing a substance, comprising a step of culturing animal cells in a serum-free medium containing soybean protein hydrolysate and yeast extract to thereby allow the animal cells to produce the substance and secrete it out of the cells and a step of isolating the substance from the serum-free medium. The substance may be a protein or peptide. The animal cells may be transformed cells into which a foreign gene has been transferred. The substance produced by and secreted out of the animal cells may be a gene product from the transferred gene, e.g., a recombinant protein or peptide. In this method, the animal cells are preferably mammalian cells, more preferably CHO cells.